Dylight 549 streptavidin

Manufactured by Vector Laboratories

Sourced in United States

DyLight 549 Streptavidin is a fluorescent labeling reagent produced by Vector Laboratories. It is a conjugate of the protein streptavidin, which has a high affinity for the molecule biotin, and the fluorescent dye DyLight 549. This reagent is designed for use in various bioanalytical and imaging applications.

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Lab products found in correlation

Donkey anti goat a488, by Thermo Fisher Scientific (1 mentions) Sm2010r, by Leica camera (1 mentions) Bisbenzimide, by Merck Group (1 mentions) 5 bromo 2 deoxy uridine labeling and detection kit 1, by Roche (1 mentions) Anti sm22α antibody, by Abcam (1 mentions) Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions) Hrp linked anti mouse igg, by Cell Signaling Technology (1 mentions) Alexa fluor 546, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Phospho akt ser473, by Cell Signaling Technology (1 mentions) β actin, by Merck Group (1 mentions) Biotinylated goat anti rabbit, by Vector Laboratories (1 mentions) Vectastain elite abc, by Vector Laboratories (1 mentions) Rabbit anti red fluorescent protein, by Abcam (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Mom basic kit, by Vector Laboratories (1 mentions) Mab5406, by Merck Group (1 mentions) Biotinylated anti streptavidin, by Vector Laboratories (1 mentions) Texas red 5 dutp, by PerkinElmer (1 mentions)

7 protocols using dylight 549 streptavidin

Cytogenetic Analysis of Festulolium Hybrids

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FISH using a telomere probe, GISH, and image analysis were performed according to Akiyama et al. (2010) with slight modifications. Genomic DNA of L. multiflorum and F. arundinacea was directly labeled with fluorescein-12-dUTP (PerkinElmer Co., Billerica, MA, USA) and Texas Red-5-dUTP (PerkinElmer Co.), respectively, by a nick translation kit (Roche, Basel, Switzerland). For the telomere probe, Arabidopsis telomere repeat sequences (Richards and Ausubel 1988 (link)) were PCR labeled with biotin-16-dUTP (Roche) and detected by Streptavidin DyLight 549 (Vector Laboratories, Burlingame, CA, USA) as the first layer of signal amplification, biotinylated anti-streptavidin (Vector Laboratories) as the second layer and again with Streptavidin DyLight 549 as the third layer. Chromosome spreads were observed under an Olympus BX61 fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan) equipped with a DP72 CCD camera (Olympus Optical Co., Ltd.). Based on the GISH results, the ratio of the Festuca-specific genomic component to the total festulolium genome (referred to as the f ratio) was analyzed using intensity values in DAPI images by Adobe Photoshop CS6 Extended (Adobe Systems Inc., San Jose, CA, USA) on a Windows 7 platform (Microsoft Corporation, Redmond, WA, USA). At least 6 chromosome spreads were analyzed to determine the f ratio in each sample.

Akiyama Y., Ueyama Y., Hamada S., Kubota A., Kato D., Yamada-Akiyama H., Takahara Y, & Fujimori M. (2016). Utilization of flow cytometry for festulolium breeding (Lolium multiflorum (2x) × Festuca arundinacea (6x)). Breeding Science, 66(2), 234-243.

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Multi-Labeling of PNNs, PV, and PCP4

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Mice were deeply anesthetized with dolethal and perfused transcardially with saline. Brains were post-fixed in 4% paraformaldehyde (two days at 4°C), then submerged in 30% sucrose solution. 30-μm-thick sections were cut on a sliding microtome (Leica SM2010R) equipped with a freezing-stage (Physitemp BFS-3MP), and placed in PB 0.1 M with saline and 0.25%Triton-X (PBST). Sections were then incubated in biotin-conjugated Wisteria floribunda agglutinin (WFA) lectin (1:1,000; Sigma, L1516), rabbit anti-PCP4 (1:500; Santa Cruz, sc-74816) and goat anti-PV antibodies (1:2,500; Swant, PVG213). The next day, floating sections were incubated for 90 min in a PBST solution containing 10% normal donkey serum, with 1:500 DyLight 549-streptavidin (Vector, SA-5549) for PNN, 1:250 donkey anti-goat A488 (ThermoFischer, A-11055) for PV, and 1:250 donkey anti-rabbit A647 (ThermoFischer, A-31573) for PCP4.

Rey C.C., Robert V., Bouisset G., Loisy M., Lopez S., Cattaud V., Lejards C., Piskorowski R.A., Rampon C., Chevaleyre V, & Verret L. (2022). Altered inhibitory function in hippocampal CA2 contributes in social memory deficits in Alzheimer’s mouse model. iScience, 25(3), 103895.

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Immunofluorescence Labeling of Glutamate

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For immunofluorescence, sections were pre-treated with 0.2% NaBH₄ in deionized water for 45 minutes to quench autofluorescence. After being rinsed in TBS with 1% MB sections were incubated with a rabbit polyclonal anti-glutamate antibody (Immunosolution, Jesmond, Australia; 1:4500) or a mouse monoclonal anti-glutamate antibody (Swant, Bellinzona, Switzerland; 1:1000) in 0.05 M TBS with 1% MB for 3 days at 4 °C.Then, sections were incubated for 1 hour at room temperature with Cy3- conjugated goat anti-rabbit immunoglobulin (Millipore, Temecula, CA; 1:100) or fluorescein conjugated goat anti-mouse immunoglobulin (Millipore; 1:100). All antibodies were diluted in TBS (pH 7.4) containing 0.2% Triton X-100 and 15% normal goat serum. Traced samples were incubated at room temperature with DyLight 549 Streptavidin (Vector; 1:1000) diluted in TBS containing 0.3% Triton X-100 for 4 h. Nuclear counterstain was carried out by immersing the slides in 0.5 μg/mL bisbenzimide (Sigma) in TBS for 5 seconds. Slides were rinsed in TBS and distilled water and mounted with Mowiol.

Fernández-López B., Barreiro-Iglesias A, & Rodicio M.C. (2016). Anatomical recovery of the spinal glutamatergic system following a complete spinal cord injury in lampreys. Scientific Reports, 6, 37786.

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Femoral Artery Wire Injury Model

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The femoral arteries of mice were subjected to wire injury. After surgery, the mice were intraperitoneally injected with 1 mg Brdu and followed with infusion of Brdu using 7-day osmotic minipump (Alzet, Cupertino, CA, USA) at a rate of 60 µg/day for 7 days. The mice were killed and perfused with 4% PFA after killing and femoral artery was dissected. The femoral artery was embedding in paraffin and 5-μm sections were cut. After blocking and antigen retrieval, the slides were incubated with indicated antibodies. BrdU was stained with a 5-Bromo-2′-deoxy-Uridine Labeling and Detection Kit I (Roche, Indianapolis, IN, USA) following the kit instruction. Anti-SM22α antibody was obtained from Abcam (catalogue number: ab10135) with 1:200 dilution. Second antibody was DyLight 549 Streptavidin (Vector laboratories, Burlingame, CA, USA).

Li Q., Fu J., Xia Y., Qi W., Ishikado A., Park K., Yokomizo H., Huang Q., Cai W., Rask-Madsen C., Kahn C.R, & King G.L. (2019). Homozygous receptors for insulin and not IGF-1 accelerate intimal hyperplasia in insulin resistance and diabetes. Nature Communications, 10, 4427.

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Quantitative Protein Analysis in Cells

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Phospho-AKT (Ser473) (RRID:AB_2315049), Cleaved caspase-3 (RRID:AB_2341188), phospho-ribosomal protein S6 (Ser235/236) (RRID:AB_916156), ribosomal protein S6 (RRID:AB_2238583), Ki67 (RRID:AB_2797703; for mouse tissue immunofluorescence), HRP-linked anti-rabbit IgG (RRID:AB_2099233) and HRP-linked anti-mouse IgG (RRID:AB_330924) were from Cell Signaling. β-Actin (RRID:AB_476692) was from Sigma-Aldrich. Ki67 (RRID:AB_443209) was from Abcam. Goat anti rabbit Alexa Fluor^® 488 (RRID:AB_2633280) and goat anti rabbit Alexa Fluor^® 546 (RRID:AB_143051) were from Invitrogen. DyLight 549 Streptavidin (RRID:AB_2336408) was from Vector Laboratories.

Kim L.M., Kim P.Y., Gebreyohannes Y.K, & Leung C.T. (2022). Sustained oncogenic signaling in the cytostatic state enables targeting of non-proliferating persistent cancer cells. Cancer research, 82(17), 3045-3057.

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MasR Expression in GABA Neurons of the BLA

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GABA reporter mice (n=3) were used for experiments determining whether the MasR is expressed on GABAergic neurons in the BLA. Tissue preparation and RNAscope in situ hybridization was performed as described in section 2.8. To quantify the frequency with which the MasR mRNA and GABA co-localize in the BLA, we used the following probes: (1) DapB, negative control, (2) Ubc, positive control, (3) Mas1, probe for MasR. To amplify the signal for tdTomato, tissue sections underwent IHC. Briefly, sections were rinsed 5 times in 50 mM potassium PBS (KPBS) and placed in a blocking solution (50 mM KPBS with 2% normal goat serum and 0.2% Triton X-100) for 1 h at room temperature. Subsequently, sections were incubated in rabbit anti-red fluorescent protein (1:200; Abcam, Cambridge, MA) in the blocking solution for 48 h at 4°C. Sections were then brought to room temperature, rinsed, and incubated for 1 h in biotinylated goat anti-rabbit (1:500; Vector Laboratories, Burlingame, CA) made in a blocking solution. Sections were then rinsed and incubated for 1 h in avidin-biotin complex (Vectastain ABC Elite, 1:500; Vector Laboratories) and, after another series of rinses, were incubated in DyLight 549 streptavidin (1:500; Vector Laboratories) for 1 h and then rinsed and cover-slipped using ProLong® Gold antifade reagent (Life technologies).

Wang L., de Kloet A.D., Pati D., Hiller H., Smith J.A., Pioquinto D.J., Ludin J.A., Oh S.P., Katovich M.J., Frazier C.J., Raizada M.K, & Krause E.G. (2016). Increasing brain angiotensin converting enzyme 2 activity decreases anxiety-like behavior in male mice by activating central Mas receptors. Neuropharmacology, 105, 114-123.

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Multistep Immunohistochemistry for Microglial and Neuronal Markers

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Tissue sections were washed three times in PBS for 8 minutes then blocked in 5% normal horse serum (NHS). Sections were then placed in a solution with primary antibody (see Table 1; anti-Iba1, made in rabbit, 1:1000, Wako Chemicals, 019–19741, RRID: AB_839504; anti-CX3CL1/fractalkine, made in goat, 1:100, R&D Systems, AF472, RRID: AB_2276839) and incubated overnight at 4°C while being agitated. For GAD67 immunohistochemistry, a monoclonal mouse anti-GAD67 antibody (clone 1G10.2, 1:100, MilliporeSigma, MAB5406, RRID: AB_2278725) was used while employing a Species-On-Species Detection Kit (Mouse on Mouse [M.O.M.] Basic Kit, Vector Laboratories, BMK-2202, RRID: AB_2336833) in order to distinguish between endogenous mouse IgG presence and the applied primary antibody. Vector M.O.M. immunodetection protocol was performed as per vendor suggestions. Species specific biotinylated secondary antibodies were used for signal amplification and visualized with a streptavidin fluorescent conjugate (DyLight 549 streptavidin, 1:200, Vector Laboratories, SA-5549, RRID: AB_2336408).

Brett C.A., Carroll J.B, & Gabriele M.L. (2021). Compromised Fractalkine Signaling Delays Microglial Occupancy of Emerging Modules in the Multisensory Midbrain. Glia, 70(4), 697-711.

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